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A Chemically Defined Synthetic Cell Capable of Growth and Replication

Nathaniel J. Gaut, Christopher Deich, Brock Cash, Tanner Hoog, Aaron E. Engelhart, Katarzyna P. Adamala

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, US

Add spudcell.jpg to this folder<br>Credit: Kate Adamala, Adamala LabDownload manuscript

SpudCell: The first synthetic cell with a complete cell cycle

Overview

Prof. Kate Adamala and her team at the University of Minnesota have built SpudCell, a cell-like system constructed entirely from known chemical components that can perform a complete cell cycle.

The system contains 36 purified enzymes, a 90,000 base pair genome spread across nine separate DNA molecules, and a lipid membrane. SpudCell is able to grow, replicate its genome, divide, and undergo selection and competition across multiple generations.

Unlike earlier work on minimal cells that carved down living cells, SpudCell is built entirely bottom-up from individually purified, non-living components. It is the first time such a system has demonstrated a complete cell cycle.

What SpudCell demonstrates

Genetically controlled feeding and growth. SpudCell grows by fusing with small &ldquo;feeder liposomes&rdquo; that deliver lipids for membrane growth plus nutrients including ribosomes, enzymes, and small molecules. Fusion happens when a protein that SpudCell makes from its own DNA locks onto the feeder&rsquo;s membrane, with the cell&rsquo;s DNA directly controlling whether it can feed, how fast it grows, and how large it becomes. Natural cells make their own nutrients through metabolism, which requires hundreds of genes encoding metabolic enzymes. By feeding externally instead, SpudCell can complete a full cell cycle with a much smaller genome.

Division without cytoskeleton. Natural cells divide using internal scaffolding called a cytoskeleton. Building a functional cytoskeleton from scratch has been a major bottleneck in synthetic cell research because it requires dozens of proteins working in coordination. SpudCell sidesteps this entirely, with proteins crowding together on the membrane surface until the mechanical stress makes the membrane split. Cells that make more of this surface protein divide more efficiently, directly coupling the genome to reproductive success.

Selection and competition. When researchers introduced a genetic change that increased production of the fusion protein, cells with that change grew faster and produced more offspring. After five generations, the faster-growing variant had outcompeted the original. Under nutrient scarcity, the advantage increased. This demonstrates selection and competition operating in a fully synthetic chemical system.

Breakdown of technical architecture

Genome organization: The 90 kbp genome is split across seven separate DNA plasmids rather than a single chromosome. Each plasmid encodes specific functions. This modular architecture allows individual functions to be modified independently. Prior analysis had speculated that a minimal genome for a living cell could be as small as 113 kbp. SpudCell&rsquo;s 90 kbp genome is smaller than this theoretical minimum.

Protein expression system: SpudCell uses the PURE (Protein Synthesis Using Recombinant Elements) system for protein expression. PURE is a defined mixture of 36 purified enzymes from E. coli bacteria, including ribosomes, that reads DNA and makes proteins. Unlike earlier approaches that used crude bacterial cell extracts, every component in PURE and its concentration is known, meaning researchers can track exactly what&rsquo;s happening inside the cell.

Chemical composition: The synthetic cells have a defined chemical composition at the time of formation, with known concentration of all components. The cells are liposomes — hollow spheres made of lipid molecules (the same fatty molecules that make up natural cell membranes). Inside each liposome is the DNA genome and the PURE protein expression system. All proteins that SpudCell needs to function are made inside the cell from its own genome.

Feeding mechanism: SpudCell uses a protein called &alpha;-hemolysin to fuse with feeder liposomes. When SpudCell makes this protein from its DNA, the protein inserts itself into the membrane and spans all the way through. A chemical tag attached to the protein sticks out from the membrane surface, binding to matching hooks on feeder liposomes and triggering fusion.

Outstanding questions and next steps

SpudCell demonstrates that many of the core processes of life can be reconstituted from fully specified, individually purified components. While there is still much more work to be done, the nanovesicle-based feeding approach provides the foundations on which we can build on. Some of the remaining challenges left to solve include:

Building ribosomes from genetic...